Reading DNA damage as foci rather than as protein: why a gamma h2ax antibody, and the gh2ax antibody and yh2ax antibody spellings catalogues use for the same reagent, reports signalling at a break rather than the break itself, what an atm antibody, an mdc1 antibody and an nbs1 antibody add about the response being assembled, how a rad51 antibody, a rad50 antibody, an mre11 antibody, a brca1 antibody, a brca2 antibody, a fancd2 antibody and a ctip antibody distinguish the repair route taken, where a blm antibody and a polq antibody mark alternative routes entirely, what an xrcc1 antibody, an ogg1 antibody, an mgmt antibody and an mlh1 antibody report about base and mismatch repair rather than breaks, how an 8 ohdg antibody or 8-ohdg antibody measures oxidative damage to the base rather than to the strand, and why a ddb1 antibody and a huwe1 antibody belong to the ubiquitin arm of the same response

Designing a damage experiment

1. Decide lesion, signal or route. An oxidised base, a signalling mark spreading from a break, and a repair factor loading are three different observations. Naming which you need selects the reagent and also the readout, because only some of them are meaningful as foci counts.
2. Count foci rather than measure intensity. The signalling mark spreads over a large chromatin domain around each break, so the informative measurement is the number of discrete foci per nucleus. Whole nucleus intensity mixes that with background and with cycle stage.
3. Control for replication. Cells in S phase carry damage marks and repair factor foci as a normal part of replication. Without a cycle marker in the same image, an apparent increase in damage can simply be more cells replicating.
4. Use a defined dose and a time course. Foci appear within minutes and resolve over hours, and the resolution curve is the informative part. A single late timepoint reports repair capacity as if it were damage.
5. Include a positive damage control on every slide. A known dose of a damaging agent on the same slide calibrates the stain and shows the reagent worked. Damage experiments without one are difficult to interpret across days.

Repair route is inferred, not observed

Loading of a recombinase or a resection factor indicates that a route is being attempted; it does not prove the break was repaired that way, and several factors participate in more than one route. Conclusions about route need more than one marker and preferably a genetic manipulation.

State inference as inference. A figure showing more recombinase foci supports a claim about pathway usage; it does not by itself demonstrate it.

Dose, time and cycle are the three axes

Every damage experiment varies along dose, time after damage and position in the cycle, and a result that fixes two of them and varies the third is interpretable. Fixing all three and comparing conditions is where most confusion enters.

Record all three with every image set. It is the metadata that makes a damage figure re-analysable rather than merely illustrative.

Common questions

Does a damage marker measure the number of breaks?

Approximately, at early timepoints and low doses, when foci are resolvable. At high dose foci merge and the count saturates, and at late timepoints the count reflects unrepaired damage rather than initial damage.

Why do untreated cells show foci?

Replication generates damage signals as a normal part of copying the genome. Staining a cycle marker alongside, and gating on non replicating cells, separates that from induced damage.

Foci or western blot?

Foci for spatial information and per cell distribution, which is what most damage questions need. A blot averages across the population and can miss a large response in a small subpopulation entirely.

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